Method for inducing synchronous condensation nucleation of materials in a short period of time

Abstract

The present invention discloses a method for inducing synchronous condensation and nucleation of materials in a short period of time. The method pre-fills the material with gas and saturates the gas; then the material is divided and placed in a freeze dryer, and the temperature and pressure are simultaneously reduced and increased to make the material in a metastable state below the crystallization point; finally, the freeze dryer pressure is reduced to below 5 psig within 10 seconds, allowing the gas in the material to quickly volatilize and induce condensation and nucleation of the material. Compared with the existing technology, the present invention has the following advantages: (1) the method of the present invention can achieve synchronous crystallization in a short period of time within a narrow specific temperature range; (2) the method can ensure uniform condensation and nucleation of the material during the freeze-drying process, without maintaining a temperature below the crystallization point for a long period of time; (3) the method can be applied to any material processing involving nucleation phase transition, such as in the production of antibody-drug conjugates.

Description

Technical Field

[0001] The invention belongs to the technical field of freeze-drying, and relates to a method for controlling uniform coagulation nucleation during the freeze-drying process, in particular to a method for inducing synchronous coagulation nucleation of materials in a short period of time. Background Art

[0002] During the current freeze-drying process, the formation of nuclei is a random event. For different samples frozen within the same batch, the timing of the appearance and initiation of coagulation is also random, resulting in asynchronous coagulation among samples within the same batch. Therefore, improvements or controls to the nucleation process are needed to ensure that the freezing of drugs or reagents in the freeze dryer occurs within a narrower temperature and time range, thereby achieving a freeze-dried product with greater uniformity from bottle to bottle. However, controlling the nucleation conditions and significantly accelerating the freeze-drying process has been a long-standing challenge for those skilled in the art.

[0003] U.S. Patent 6,682,524 discloses a method for vacuum-induced surface freezing. Bottles containing an aqueous solution are loaded onto a temperature-controlled rack in a freeze dryer and maintained at an initial temperature below 10°C. The freeze drying chamber is then evacuated to a near-vacuum state, causing the surface of the aqueous solution to freeze to a depth of several millimeters. Releasing the vacuum and lowering the rack temperature below the freezing point of the solution subsequently allow ice crystals to grow from the pre-frozen surface layer through the remaining portion of the solution. A major drawback of this method is that the process of evacuating the liquid to release gases within the liquid can easily cause the solution to boil violently or degas.

[0004] Chinese patent CN101379356B discloses a method for inducing nucleation of a material, wherein the material is brought to a state below the thermodynamic freezing point and a metastable state in a freeze-drying chamber at a pressure of 7 to 50 psig; and within 40 seconds or less, the pressure in the freeze-drying chamber near the material is rapidly reduced to induce frozen nucleation in the material. Specifically, the method is to start decompressing the freeze-drying chamber when the material reaches the desired nucleation temperature, or to start decompressing the freeze-drying chamber at a desired time after the material temperature is below the thermodynamic freezing point. Paragraph

[0045] of the specification discloses that it is preferred to place the material in the freeze-drying chamber and allow the temperature, pressure and gas atmosphere of the freeze-drying chamber to be controlled. The role of the gas atmosphere is, on the one hand, to control the pressure in the chamber, and on the other hand, to serve as a temperature heat transfer fluid for the material in each bottle or container. The advantage of this prior art is that it avoids the solution from boiling violently or degassing due to vacuuming, thereby improving the randomness of controlling nucleation during the freeze-drying process caused by violent boiling or degassing of the solution to a certain extent. However, this method, which introduces gas into the freeze-drying chamber, cannot guarantee that the gas reaches saturation in every bottle or container, and therefore cannot guarantee uniform nucleation. Furthermore, the method of cooling first and then decompressing the air takes a long time. As mentioned in paragraph

[0255] of the specification, this process requires the freeze-drying rack to be maintained at -14°C for 16 hours. These deficiencies all affect the nucleation rate.

[0005] Therefore, although gas (such as nitrogen) is added to the solution material in the prior art, it is all added into the freeze dryer or drying chamber, so it is impossible to ensure that each bottle or container reaches saturation. The method of first cooling and then reducing the pressure in the freeze dryer or drying chamber requires maintaining a temperature below the crystallization point for a long time to achieve instantaneous synchronous crystallization caused by the gas volatilization process. The disadvantage of the above technology is that it cannot ensure uniform condensation and nucleation during the freeze-drying process, and it takes a long time, thereby affecting the nucleation rate.

[0006] The existing technology still seldom applies controlled nucleation technology to actual production. Simultaneous cooling and pressure increase require very high equipment requirements, and currently, there are few mature devices capable of this function. Therefore, the focus of technical personnel is largely on equipment development. Furthermore, existing technology often uses existing instruments to explore the range of material nucleation temperature and pressure reduction rate. While the research goal is to achieve nucleation, this fails to address the problems of long operation times and uncontrollable nucleation rates in actual operation. Summary of the Invention

[0007] Technical Problem to Be Solved: To overcome the shortcomings of existing technologies, a process is needed to ensure uniform nucleation of materials during the freeze-drying process while minimizing time. Specifically, the process involves achieving gas saturation within the material within a short period of time under specific temperature conditions, and then utilizing a sudden change in pressure to achieve instantaneous synchronous crystallization. To address this issue, the present invention provides a method for inducing synchronous nucleation of materials within a short period of time.

[0008] Technical solution: A method for inducing simultaneous condensation and nucleation of materials in a short period of time, comprising the following steps:

[0009] Pre-fill the material with gas and saturate it with gas;

[0010] The material is packaged and placed in a freeze dryer, and the temperature is cooled and the pressure is increased simultaneously. The temperature in the freeze dryer is controlled at -2 to -8°C and the pressure is 25 to 28 psig, so that the material is in a metastable state below the crystallization point.

[0011] The freeze dryer pressure is reduced to 2-5 psig within 10 seconds to allow the gas in the material to evaporate rapidly, thereby inducing the material to condense into nuclei.

[0012] The metastable state described above refers to a material's state below its phase transition temperature. Metastable states are unstable and transient, but relatively long-lived, states in chemical or biological systems. In the absence of any changes in the material or its environment, a metastable material will eventually transition from its non-equilibrium state to its equilibrium state.

[0013] Preferably, the material can be a pure substance, gas, suspension, gel, liquid, solution, mixture, or component of a solution or mixture. Specifically, it includes biopharmaceutical materials, chemical pharmaceutical materials, live or attenuated viruses, nucleic acids, monoclonal or polyclonal antibodies, proteins, peptides, or non-peptide analogs. The method of the present invention requires dissolving the above materials in water, and the aqueous solution is pre-treated with a filter membrane to remove particles that interfere with nucleation, promote the formation of a uniform ice crystal structure, and reduce freeze-drying time.

[0014] Preferably, the material is microfiltered before being pre-filled with gas.

[0015] Preferably, the gas pre-filled in the material includes at least one of nitrogen, argon, helium, neon, xenon, and krypton.

[0016] Preferably, the purity of the gas pre-filled in the material is above 99.99%, and the gas flow rate is 0.5 to 1 L / min.

[0017] Preferably, the temperature condition for the pre-filled gas in the material to reach saturation is 18-25°C.

[0018] Preferably, the freeze dryer is pre-cooled to 2-5°C before the subpackaged materials are placed in it.

[0019] Preferably, after being placed in a freeze dryer, the temperature is lowered from the pre-cooling temperature to -2 to -8°C within 10 to 20 minutes, and the pressure is increased from normal pressure to 25 to 28 psig, and maintained for 10 to 30 minutes.

[0020] Preferably, the pressurization process is to introduce at least one gas selected from nitrogen, argon, helium, neon, xenon, and krypton into the freeze dryer.

[0021] Preferably, the temperature inside the freeze dryer is maintained at -2 to -8°C during the depressurization process. Within this temperature range, rapid depressurization causes the previously elevated pressure inside the freeze dryer to be rapidly reduced by the outflow of the gaseous atmosphere, and rapid depressurization can induce nucleation. Specifically, rapid depressurization rapidly reduces the solubility of the gas previously saturated with solution within the material, and the rapid release of gas from the metastable solution triggers a phase transition to nucleation.

[0022] In principle, the method of the present invention can be applied to any material processing involving nucleation phase transition, such as liquid freezing, ice crystallization of drug aqueous solutions, protein crystallization, food freezing, freeze concentration, etc. However, the most urgent application for the applicant is to improve the current drug freeze-drying method. For example, in the industrial freeze dryer of a pharmaceutical manufacturer, there are batches of vials containing drug products that need to be frozen and dried. The conventional practice in production is to cool the temperature in the freeze dryer to a very low level to ensure that all the drugs in the vials are frozen, but the drugs in each bottle freeze randomly within the temperature range below the freezing point because the nucleation process is uncontrolled. The specific application range is such as in the production process of antibody-drug conjugates, to achieve synchronous coagulation nucleation in a short time.

[0023] In view of this, the principle of the present invention is that the nucleation temperature is proportional to the size of the ice nucleus. The higher the nucleation temperature, the larger the ice nucleus, the coarser the pores left after the ice crystals sublime, the smaller the sublimation resistance, and the faster the sublimation rate. The present invention raises the temperature in the freeze dryer to -2°C, and makes the gas in the material reach saturation in a short time, and then uses the pressure mutation to achieve instantaneous synchronous crystallization. The above method makes the nucleation temperature of the liquid in different containers in the same freeze dryer more uniform, and the nucleation temperature is increased, which is conducive to the generation of larger ice nucleus size and improves the drying efficiency of the material.

[0024] Beneficial effects: (1) The method of the present invention can achieve synchronous crystallization in a short time within a narrow specific temperature range; (2) The method can ensure the uniform condensation and nucleation of the material during the freeze-drying process, and there is no need to maintain a certain temperature below the crystallization point for a long period of time; (3) The method of the present invention can increase the nucleation temperature of the material to -2°C, which is conducive to generating larger ice nucleus size, thereby helping to increase the drying rate of the material. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the placement of samples (such as vials) in the freeze dryer in the embodiment;

[0026] Figure 2 The pressure and temperature-time curves of the present invention method using reduced pressure to control nucleation for 30 minutes, where 1 is the chamber pressure in psig, 2 is the plate temperature in °C, 3 is the product temperature in °C, the vertical axis is temperature, and the horizontal axis is time;

[0027] Figure 3 This is the pressure and temperature-time curve after the method of the present invention utilizes reduced pressure to control nucleation and maintain it for 10 minutes, where 1 is the cavity pressure psig, 2 is the plate temperature °C, 3 is the product temperature °C, the vertical axis is temperature, and the horizontal axis is time. DETAILED DESCRIPTION

[0028] The following examples further illustrate the present invention but are not to be construed as limiting the present invention. Modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention are intended to fall within the scope of the present invention. Unless otherwise specified, the techniques used in the examples are conventional means well known to those skilled in the art.

[0029] The freeze drying process described in this paper was carried out in an SP Scientific freeze dryer (model SP Hull 4.0). A freeze dryer partition is provided inside the freeze dryer, and a vial containing a drug solution is placed on the partition. A vent valve is provided on the rear side of the machine, and a pipeline is connected to the vent valve for releasing gas or introducing gas to increase the pressure. The drug solution is prepared in a biological safety cabinet, and in particular, the cabinet is filled with gas to reach a saturated state. In this example, nitrogen is used as the gas and 5% sucrose solution as the material. The specific process of the method is as follows:

[0030] Example 1 Pressurization maintenance time 30min

[0031] S1. Prepare 500 mL of 5% sucrose solution in a PC bottle, filter it through a 0.22 μm filter membrane, and divide it into two bottles of 250 mL each. Place them in a biosafety cabinet. Maintain the temperature in the biosafety cabinet between 18 and 25°C. Connect one bottle of solution to a glass nozzle connected to a nitrogen line with a purity of 99.99% or higher and pass it into the bottom of the liquid.

[0032] S2. Close the pressure control valve on the nitrogen tank, open the main valve of the nitrogen tank, then slowly unscrew the pressure control valve and control the nitrogen flow rate between 0.5 and 1 L / min. At this flow rate, the continuous filling of nitrogen can be guaranteed while the liquid medicine will not be flushed out by the airflow. The filling process lasts for 30 minutes to make the nitrogen dissolved in the liquid medicine reach saturation.

[0033] S3. Under the condition of 18-25℃, in a biological safety cabinet, divide the solution dissolved with saturated nitrogen and the solution without inflation into 15 bottles of penicillin, with a filling volume of 5mL. Place them on the partition of the freeze dryer that has been pre-cooled to 5℃. The inflation and non-inflation sample solutions should be symmetrical and arranged in a V shape, as shown in the figure. Figure 1 Close the freeze dryer door as shown.

[0034] S4. Set the freeze dryer program so that the freeze dryer shelf temperature drops to -5°C within 10 minutes and is maintained at this temperature for 30 minutes. During this time, the entire liquid medicine drops to -4°C. Simultaneously, nitrogen is introduced through the vent valve to raise the pressure in the freeze dryer shelf chamber to 28 psig. At this point, the liquid medicine is in a metastable state below the crystallization point, but has not yet reached the supercooling point, so no crystallization occurs.

[0035] S5. When the diaphragm temperature is -5°C, open the vent valve to connect it to the atmosphere and reduce the pressure to 2 psig in <10 seconds. When the pressure is released instantaneously, the nitrogen dissolved in the liquid will evaporate during the pressure release process, causing the movement of water molecules in the solution to intensify, thereby reaching the critical size for nucleation and causing crystallization.

[0036] The high pressure was maintained for 30 min to control the nucleation results. See Table 1

[0037] Table 1 High pressure maintained for 30 min to control nucleation results

[0038]

[0039] from Figure 2 It can be seen that the sample solution first dropped to approximately -4°C, below its nucleation temperature, and was maintained at 28 psig for 30 minutes. Upon pressure release, the solution temperature simultaneously abruptly rose to 0°C, indicating that it had transformed from water to ice and successfully nucleated. The nucleation process was completed within a few seconds. As shown in Table 1, the nucleation rate for both solutions was 100%. This is because the high pressure was maintained for a long time, 30 minutes, which allowed the solution temperature to drop to -4°C, below its crystallization point. Furthermore, maintaining 28 psig for 30 minutes was sufficient to saturate the solution with nitrogen in both the pre-charged and non-pre-charged vials. Therefore, there was no significant difference in the nucleation rate between the two solutions.

[0040] Example 2: Reduce the pressurization time to 10 minutes

[0041] S1. Prepare 500 mL of 5% sucrose solution in a PC bottle, filter it through a 0.22 μm filter membrane, and divide it into two bottles of 250 mL each. Place them in a biosafety cabinet. Maintain the temperature in the biosafety cabinet between 18 and 25°C. Connect one bottle of solution to a glass nozzle connected to a nitrogen line with a purity of 99.99% or higher and pass it into the bottom of the liquid.

[0042] S2. Close the pressure control valve on the nitrogen tank, open the main valve of the nitrogen tank, then slowly unscrew the pressure control valve and control the nitrogen flow rate between 0.5 and 1 L / min. At this flow rate, the continuous filling of nitrogen can be guaranteed while the liquid medicine will not be flushed out by the airflow. The filling process lasts for 30 minutes to make the nitrogen dissolved in the liquid medicine reach saturation.

[0043] S3. Under the condition of 18-25℃, in a biological safety cabinet, divide the solution dissolved with saturated nitrogen and the solution without inflation into 15 bottles of penicillin, with a filling volume of 5mL. Place them on the partition of the freeze dryer that has been pre-cooled to 5℃. The inflation and non-inflation sample solutions should be symmetrical and arranged in a V shape, as shown in the figure. Figure 1 Close the freeze dryer door as shown.

[0044] S4. Set the freeze dryer program so that the freeze dryer shelf temperature drops to -5°C within 10 minutes and is maintained for 10 minutes. During this time, the drug solution drops to -2°C. Simultaneously, nitrogen is introduced through the vent valve to raise the pressure in the freeze dryer shelf chamber to 28 psig. At this point, the drug solution is in a metastable state below the crystallization point, but has not reached the supercooling point, so no crystallization occurs.

[0045] S5. When the diaphragm temperature is -5°C, open the vent valve to allow it to vent to the atmosphere and reduce the pressure to 2 psig in <10 seconds. Maintain the high pressure for 10 minutes to control the nucleation results. See Table 2

[0046] Table 2 High pressure maintained for 10 min to control nucleation results

[0047]

[0048]

[0049] After the plate layer dropped to -5℃, it was kept under 28psig high pressure for 10min. During this period, the sample liquid dropped to about -2℃, slightly lower than the nucleation temperature. Figure 3 It can be seen that during pressure release, the temperature of the temperature probe changed slightly, rising to around 0°C, indicating successful nucleation. However, because the high pressure was maintained for only 10 minutes, the ungassed sample was not fully saturated within 10 minutes, resulting in incomplete nucleation. The nucleated sample also showed incomplete nucleation. The pre-nitrogen-filled solution, however, was saturated with dissolved nitrogen prior to packaging. When all vials were cooled to -2°C in the freeze dryer, reaching the crystallization point, and the pressure was released from 28 psig to 2 psig, all pre-gassed solutions nucleated within a very short period of time. The results are shown in Table 2.

[0050] In summary, the pre-gassing method can ensure the uniform condensation and nucleation of the material during the freeze-drying process, and achieve synchronous crystallization in a short time within a narrow specific temperature range, without the need to maintain a certain temperature below the crystallization point for a long period of time; pre-gassing can also increase the nucleation temperature of the sample liquid to -2°C, which is conducive to the generation of larger ice nucleus size, thereby helping to increase the drying rate of the drug solution.

Claims

1. A method for inducing simultaneous condensation and nucleation of materials in a short period of time, characterized in that: Including steps: Pre-fill the material with gas and saturate it with gas; The material is packaged and placed in a freeze dryer, and the temperature is cooled and the pressure is increased simultaneously. The temperature in the freeze dryer is controlled at -2~-8℃ and the pressure is 25~28psig, so that the material is in a metastable state below the crystallization point. Reduce the freeze dryer pressure to 2-5 psig within 10 seconds to allow the gas in the material to evaporate quickly and induce the material to condense into nuclei; The gas pre-filled in the material includes at least one of nitrogen, argon, helium, neon, xenon, and krypton; The purity of the gas pre-filled in the material is above 99.99%, and the gas flow rate is 0.5~1L / min; Before placing the subpackaged materials into the freeze dryer, pre-cool them to 2-5°C; The material includes a biopharmaceutical material or a chemical drug material; During the depressurization process, the temperature inside the freeze dryer was maintained at -2~-8℃.

2. The method for inducing simultaneous condensation and nucleation of materials in a short period of time according to claim 1, characterized in that: The material is microfiltered prior to pre-filling with gas.

3. The method for inducing simultaneous condensation and nucleation of materials in a short period of time according to claim 1, characterized in that: The temperature condition for the pre-filled gas in the material to reach saturation is 18~25℃.

4. The method for inducing simultaneous condensation and nucleation of materials in a short period of time according to claim 1, characterized in that: The freeze dryer is cooled from the pre-cooling temperature to -2~-8℃ within 10~20 minutes, and the pressure is increased from normal pressure to 25~28 psig, and maintained for 10~30 minutes.

5. The method for inducing simultaneous condensation and nucleation of materials in a short period of time according to claim 1, characterized in that: The pressurization process is to introduce at least one gas selected from nitrogen, argon, helium, neon, xenon and krypton into the freeze dryer.

Images